The creation of tubular networks is essential to the production of such organs as the lungs, the kidneys, and the vascular system. The Drosophila tracheal system provides an excellent model system in which the formation of tubular networks can be studied using powerful genetic, molecular, and genomic tools. Previous work has shown that there is a high degree of conservation between flies and vertebrates in the molecular pathways used to mediated tube branching. This proposal focuses on another aspect of tracheal network formation, branch fusion. Growing branches must identify and move towards the appropriate fusion partner, then align and form an appropriate connection. These fusion events are essential to the formation of an interconnected network, and remain poorly understood at the cellular and molecular level. Fusion events are mediated by a single cell, called a fusion cell, found at the ends of growing branches. To identify the genes that act during branch fusion, fusion cells will be isolated from embryos by whole animal cell sorting (WACS), and cDNAs made from these cells will be used to probe DNA microarrays. Comparison of genes expressed in fusion cells to genes expressed in other tracheal cells allow identification of fusion cell- specific genes. The expression pattern of these candidate genes will be verified by in situ hybridization, and the loss of function phenotypes of these genes will be determined by RNA interference (RNAi) experiments. Genes will be categorized and ordered into pathways, laying the foundation for a detailed molecular understanding of each cellular step in the fusion process.